In microscopy of live eukaryotic and prokaryotic cell cultures, including oocytes and embryos during, e.g., IVF treatment, it is generally desirable to reduce stress imposed on the cell cultures during handling thereof.
The diameter of early human embryos is about ⅛ mm (approximately 120 μm) with a density slightly higher than their growth medium. Positioning embryos accurately in media droplets is difficult, and handling may easily disturb their position. When applying Embryo Transfer (ET) techniques, such as IVF (In Vitro Fertilization) and related techniques, in vitro culturing of the developing embryo is carried out for a period of days before transfer of selected embryos back to uterus of the recipient patient. Even under ideal growth conditions, selection criteria are needed as a tool to choose the most viable embryos for transfer as most embryos have genetic defects (e.g. aneuploidy) that prevent them from developing to healthy infants. Assessment of the viability of an embryo will determine the embryos' suitability for transfer. In traditional IVF, embryo assessment is limited to a more or less subjective grading based on morphological criteria. As the embryo development is a dynamic and gradual process it is most readily and comprehensively evaluated by a succession of images such as those provided by Time-Lapse (TL) microscopy. Automation is essential when capturing images at defined time intervals of numerous growing embryos and is a prerequisite for clinical use of TL imaging for embryo viability assessment. At a practical level, precise positioning for microscopy facilitates assessment of the viability of an embryo based on automated time-lapse imaging.
Therefore, a need exists for a fast, simple and non-disturbing method, system and device for facilitating and automating morphological evaluation.
International patent publication No. WO 2009/003487 discloses a device for use during monitoring and/or culturing of microscopic objects. The device disclosed therein addresses issues related to providing stable incubation conditions, and to facilitating handling of the objects, including automated handling.
A microscope is normally used for optically monitoring cell cultures, such as embryos. Digital image acquisition and analysis equipment is typically applied to assist a human examiner in deriving appropriate information as needed from the acquired microscope images. In conventional microscopy applications, such as those used in IVF, a certain distance usually exists between an optical lens of a microscope and the cell culture to be monitored, the distance being given by the thickness of a tray accommodating the cell culture, insulating and heat conducting elements providing a thermostatic environment and sealing elements to maintain a controlled atmosphere and air gaps between the aforementioned elements to allow for mechanical movement and exchange of the cell cultures being monitored.
The quality of the captured microscope images evidently plays a role for the quality of the analysis that can be made on the basis of the images. One way of increasing image quality is to increase the pixel resolution of the digital camera equipment, which captures the images. However, increasing camera resolution beyond the resolution of the optical system will not provide additional detail but only magnify the image reproducing the blurry outlines of the visible components.
The resolution of the employed lens system can be described by the required distance between two tiny objects for them to be perceived as separate objects and not part of the same elongated object (cf. descriptions of optical systems, Airy disk etc.): If two objects imaged by an optical system are separated by an angle small enough that their Airy disks on the optical systems detector (i.e. camera) start overlapping, the objects can not be clearly separated any more in the image, and they start blurring together. Two objects are said to be just resolved when the maximum of the first Airy pattern falls on top of the first minimum of the second Airy pattern (the Rayleigh criterion). Therefore the smallest separation two objects can have before they significantly blur together is approximated by the size of the Airy disk:h=0.61λ/NAwhere λ is the wavelength of the light and NA is the numerical aperture given by:NA=n*sin(e)
Where n is the refractive index and θ is the half-angle of the maximum light cone collected by the optical system.
One way of increasing the optical resolution (i.e. decreasing h) would be to reduce the wavelength of the light used (e.g. change from red to green or preferably blue light or even most preferably to UV light). However, short wavelength light has higher energy and has been shown to be far more phototoxic to living organisms than long wavelength light. For clinical applications it is thus advisable to use only long wavelength red light to minimize any potential damage to living organisms.
Another way of increasing image quality (i.e. decreasing h) is to increase the numerical aperture of the optical system. This can be accomplished by increasing the refractive index (e.g. by liquid immersion microscopy) or by increasing the half-angle of the light cone collected by the microscope objective. Liquid immersion is impractical in an automated system with moving parts that mechanically change between acquiring images of different cells/embryos. Even in a stationary system cleaning and handling is more complicated when using liquid immersion and there is a larger potential for contamination.
Increasing the acceptance angle for the light cone of the collected light is usually accomplished by positioning the objective closer to the investigated object while increasing the magnification of the objective. High magnification objectives with high numerical aperture thus require close proximity between the observed object and the position of the microscope lens. However, it is not always possible to place the objective close to the living cells for instance if the observed object must be mechanically exchanged with other similar objects and if the object must be in a protected stable environment (e.g. in a thermostatic holder with direct heat transfer to the culture vessel). Cultivation systems that maintain a thermostatic environment and a controlled atmosphere optimized for embryo development often require a minimal distance/separation between the living cells and the microscope objective as mentioned above.
Increasing the diameter of the optical lens to increase the acceptance angle of incident light is generally prohibitively expensive. The numerical aperture of an optical system based on standard microscope elements (objective and tubal lenses and camera lenses) can thus not be increased infinitely, and the requirement to maintain a stable environment for embryo development/cell culture may further limit the optical resolution that can be achieved. The optical resolution and hence image quality is therefore limited.